Adenosine deaminase (ADA) is an enzyme of purine nucleoside metabolism that is present in virtually all human tissues. The highest levels are found in the thymus and are subject to developmental regulation within this tissue. Individuals lacking ADA suffer from a genetic disorder termed severe combined immunodeficiency disease characterized by a lack of B- and T-lymphocytes. Thus, the genetic evidence indicates that ADA is essential for development of the immune system. To address a number of questions regarding ADA gene structure, the regulation of ADA gene expression, and the molecular basis of ADA deficiency, we propose to clone the ADA gene from cell lines we have isolated which have a 6,000-fold amplification of the ADA gene. From these cloned sequences we propose to place ADA minigenes in eukaryotic expression vectors for use in DNA mediated gene transfer into ADA deficient cells. These studies will serve as a rational beginning for the use of cloned ADA genes in gene replacement therapy. For a number of reasons human ADA deficiency is an ideal model genetic disorder for which to consider gene replacement therapy. The proposed research will use recombinant DNA technology to analyze a specific human genetic disorder. The information gained will be relevant not only for this one genetic disease but to all human genetic disorders that become manifest in bone marrow derived cells.